Targeting Catalytic Residues of TKTL1: A Multi‐Approach Study With Thiamine Analogs Combining QSAR, Pharmacophore Modeling, DFT, and Molecular Dynamics

ABSTRACT The TKTL1 protein, which links glycolysis and the pentose phosphate pathway, is a promising target for inhibiting ATP synthesis in cancer cells. Its active site residues (Ser44, Ser49, Ser53, Lys84, Glu128, and Lys218) facilitate protonation and Schiff base reactions critical for catalytic activity. Despite prior inhibitor failures, this study rationalized the use of thiamine analogs to disrupt these reactions. The five‐point pharmacophore hypothesis, “AADHR”, was highly predictive and prolific, resulting in a pool of compounds with high binding affinity. The hypothesis was statistically significant, with a correlation coefficient of r 2 = 0.73 and a cross‐validation correlation coefficient of q 2 = 0.55. The external validation result displays the significance of the model, with r 2 (o) of 0.99 and r 2 (m) of 0.51. Quantum mechanical calculations were applied to the top five screened compounds, which inferred the deformation of the chemical reaction of the inhibitory mechanism of the TKTL1 protein. Molecular dynamics and MM/PBSA analyses revealed stable RMSD and favorable binding‐free energies for TKTL1–ZINC95911396, TKTL1–Spec‐AH‐034, and TKTL1–MayBridge‐BTB07677, indicating that dynamic stability can compensate for stronger binding in determining inhibitory potential. Overall, our findings identify a promising TKTL1 inhibitor that strongly correlates with existing experimental data, highlighting the potential of novel compounds to inhibit TKTL1 activity in cancer cells.